Lipid Nanovesicle Platforms for Hepatocellular Carcinoma Precision Medicine Therapeutics: Progress and Perspectives

ABSTRACT Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related mortality globally. HCC is highly heterogenous with diverse etiologies leading to different driver mutations potentiating unique tumor immune microenvironments. Current therapeutic options, including immune checkpoint inhibitors and combinations, have achieved limited objective response rates for the majority of patients. Thus, a precision medicine approach is needed to tailor specific treatment options for molecular subsets of HCC patients. Lipid nanovesicle platforms, either liposome- (synthetic) or extracellular vesicle (natural)-derived present are improved drug delivery vehicles which may be modified to contain specific cargos for targeting specific tumor sites, with a natural affinity for liver with limited toxicity. This mini-review provides updates on the applications of novel lipid nanovesicle-based therapeutics for HCC precision medicine and the challenges associated with translating this therapeutic subclass from preclinical models to the clinic.


Introduction
Hepatocellular Carcinoma (HCC) is a growing global public health burden.HCC is the sixth most common cancer globally, with > 900,000 cases each year, and the third highest in cancerrelated mortality, with > 800,000 deaths each year. 1 HCC typically follows a sequalae of chronic liver disease, with the main etiologies including Hepatitis B and C virus (HBV & HCV) infection, alcohol-related disease, steatotic liver diseases (SLD) (e.g., metabolic dysfunction associated SLD [MASLD], diabetes mellitus, obesity), and toxin exposure (e.g., cigarette smoke, aflatoxin, liver fluke). 2 HCC has a dismal prognosis with a 5-year overall survival (OS) rate of ~ 15-20% and <18 months median survival with current therapeutic paradigms. 3Very few patients are diagnosed at early stages where surgical resection/transplantation is feasible and nearly curative. 4In fact, the vast majority of patients are diagnosed with advanced disease, limiting their options to systemic agents, including tyrosine kinase inhibitors (TKIs) and, more recently, immune checkpoint inhibitors (ICIs).][7] Therefore, novel targeted therapies used in conjunction with immunotherapy, in a precisionmedicine based approach, may overcome HCC therapeutic resistance to ICIs in molecular subsets of patients.Lipid nanovesicle platforms, either liposome-(synthetic) or extracellular vesicle (natural)-derived, have demonstrated promise as drug delivery vehicles to the liver and for targeted cancer agents.These nanocarriers are ideal drug delivery vehicles which may be functionalized to harbor specific cargo molecules and "home" to specific tumor sites, with native affinity for liver with limited toxicity. 8,9This mini-review discusses HCC molecular subclasses and current treatment paradigms, along with applications of novel lipid nanovesicle-based therapeutics for HCC precision medicine with a focus on naturally-derived nanovesicle formulations, and the challenges associated with translating this new therapeutic subclass from preclinical models to the clinic.
The two main molecular classification systems proposed are the G1-G6 system by Boyault et al. 13 and the S1-S3 subgroups by Hoshida et al. 14 Briefly, G1-G3 and S1-S2 subclasses represent proliferative/ poorly differentiated tumors associated with chromosomal instability, high HBV viral load, and TP53 mutations, while G5-G6 and S3 subclasses represent non-proliferative/well-differentiated tumors associated with chromosomal stability, alcohol/HCV/ NASH-driven HCC, and CTNNB1 mutations. 13,14ore recently, HCC can be classified into inflamed (Hoshida S1-S2 subgroups) or non-inflamed (Hoshida S3) subgroups. 14,15The inflamed class of HCC (~25% of patients) demonstrates increased expression of gene signatures related to immune infiltration (i.e., cytotoxic T cells, tertiary lymphoid structures [TLS], IFN alpha and gamma signaling, and chemokines CXCL9, CXCL10), high immune checkpoint immunohistochemical expression, CTNNB1-mutated depleted, and enrichment of amplification in q13 locus (CCND1, FGF19). 15ditionally, the inflamed class can be further subdivided into either immune-active or immuneexhausted, with the immune-active subclass representing high adaptive immunity gene expression with improved survival and reduced rates of recurrence.The immune exhausted subclass demonstrates activated stroma and immunosuppressive gene set signatures. 16Overall, these classification systems illustrate how tumor genetics drive both tumoral heterogeneity and specific tumor microenvironments, which may be differentially susceptible to various systemic agents, and thus, may require tailored treatment options for patients informed by tissue and/or liquid biopsy. 17

Current treatment modalities and patient selection
For advanced HCC, current standard of care has shifted from the use of TKIs toward ICIs in the last decade.ICIs are monoclonal antibodies which block the interaction between immune checkpoint molecules (e.g., programmed death-ligand 1 [PDL1] on tumor cells interacting with programmed cell death protein 1 [PD1] on T cells) potentiating cytotoxic CD8+ T cell mediated tumor cell killing. 18The IMbrave 150 trial demonstrated 19.2 months median survival with atezolizumab (anti-PDL1 antibody) plus bevacizumab (anti-VEGF antibody) compared to 13.4 months median survival with sorafenib (TKI). 19Also, the HIMALAYA trial demonstrated 16.4 months median survival with the ICI combination of tremelimumab (anti-CTLA4 antibody) plus durvalumab (anti-PDL1 antibody) compared to 13.7 months median survival with sorafenib. 7Moreover, the CARES-310 trial demonstrated 22.1 months median survival with camrelizumab (anti-PD1 antibody) plus the VEGFR2-targeted TKI rivoceranib compared to 15.2 months median survival with sorafenib. 20However, despite the improved OS in ICI treated patients, response rates overall remain relatively low with only 25-30% of patients achieving objective response rates (ORR).Low ORRs are poorly understood but have been linked to patient tumor microenvironments with low tumorinfiltrating effector T lymphocyte density, high regulatory T cell density, and high expression of oncofetal genes. 21Thus, to improve these response rates, an individualized treatment approach is warranted to guide therapeutic selection based on underlying genetic alterations.This may be aided by tissue or liquid biopsy for key drivers of HCC tumorigenesis. 17However, an improved understanding of which genetic drivers influence the immune microenvironment resistant to ICI response is warranted for screening, along with needing an expanded arsenal of drugs targeting these underlying pathways to be used in conjunction with ICIs.
On a molecular basis, the Wnt/β-catenin pathway has been the most prominently studied pathway to evaluate ICI resistance, yet controversy remains whether all mutations in the pathway decrease immune infiltration to the same degree, and thus ICI resistance. 16,21,22 Mreover, despite studies demonstrating the feasibility of prospective tissue genotyping to identify clinically actionable driver mutations, very few patients receive personalized therapeutic intervention. 10The major driver mutations in HCC are currently not actionable 23; therefore, efforts should be made to identify and stratify patients which may respond to current druggable targets, including FGF19/FGFR4, VEGF, TSC1/2, and MET inhibitors. 24,25Although none of these targets have shown clinical responses, these molecular events may be co-occurring in the background of strong drivers (e.g., TP53, CTNNB1), and thus a combination of therapeutics may need to be eventually employed.Thus, further studies are needed in clinically relevant animal models to determine the differential response of ICIs in combination with targeted therapy approaches in unique molecular subsets of HCC.

Synthetic lipid nanovesicle drug delivery platforms for HCC
Synthetic lipid nanovesicles have conventionally been nanoliposome-based formulations containing distinct molecular entities, including either RNA interference (RNAi) technologies or chemotherapeutic drugs.Nanoliposomes typically size range between 10 nm to 200 nm in diameter and are composed of a phospholipid bilayer with or without cholesterol, resulting in an aqueous interior and an outer hydrophobic exterior. 26The main types of nanoliposomes include small unilamellar vesicles (<100 nm), large unilamellar (>100 nm), and multilamellar vesicles (>500 nm), with the former two more typically used for nanomedicine applications. 27Excellent reviews elsewhere discuss preparation methodologies (e.g., reverse-phase evaporation, freeze-thaw method, vaporization technique, and others) of nanoliposome formulations. 28,29Briefly, the phospholipid characteristics (e.g., degrees of unsaturation, quantity of fatty acid moieties, and others) and the number of cholesterol molecules can affect the membrane configuration. 30,31Further modifications to the nanoliposomal structure include the addition of either polyethylene glycol 32 or surface ligands, 33 which avoids host immune system elimination and improves cellular targeting, respectively.For cellular uptake, nanovesicles are internalized typically through endocytosis or phagocytosis, with nanovesicle structure influencing which mechanistic process. 34fficient perfusion of the liver through its dual blood supply mediates optimal delivery, and lipid nanovesicle uptake is augmented due to its fenestrated endothelium.Additionally, opsonization by ApoE facilitates low-density lipoprotein (LDL) receptor (LDLR)-mediated uptake into hepatocytes ("endogenous targeting"), while engineering N-acetylgalactosamine (GalNAc)-PEG-lipid on the nanovesicle surface can target the asialoglycoprotein receptor (ASGPR) on hepatocytes ("exogenous targeting"), with both options providing efficient delivery to the liver. 35,36This well-characterized ApoE-LDLR endogenous hepatocyte targeting mechanism is the route by which Patisiran, the first FDA approved siRNA-based drug, facilitates its endorgan targeting to the liver and mechanism of action. 37The remainder of this section will discuss the applications of nanoliposomes as targeted drug delivery vehicles in various preclinical models of HCC as potential precision medicine therapeutic platforms (Figure 1).
The realization of using lipid nanovesicles as a targeted therapy delivery vehicle for liver cancer in humans was first achieved in 2013 by Tabernero and colleagues in their phase I study.This lipid nanovesicle (ALN-VSP) encapsulated siRNAs targeting vascular endothelial growth factor (VEGF) and kinesin spindle protein (KSP) to treat patients with liver metastases. 38Tumor regression was achieved in nearly 50% of the patients in the trial.These results, demonstrating the safety, tolerability, ability to achieve target downregulation in the liver, and short-term clinical responses underscore the importance and potential of using lipid nanovesicles for HCC therapy.
Nanoliposomes encapsulating RNA interference (RNAi) platforms, such as small interfering RNAs (siRNAs), microRNAs (miRNAs), or messenger RNAs (mRNAs) have been administered as drug delivery systems in preclinical models of HCC with considerable success in terms of safety, tolerability, and treatment response.Various groups have attempted to use RNAi to either target oncogenic genes involved in cell cycle regulation and cell proliferation/death pathways, or directly inhibit driver mutations deemed to be traditionally "undruggable."0][41][42] An example of directly targeting oncogenic factors is illustrated by work from Younis colleagues where they encapsulated both a siRNA to midkine (MK; a gene involved in many cellular pathways including apoptosis and angiogenesis 43 and the chemotherapeutic, sorafenib, into a nanoliposome functionalized to contain 3 components: 1) SP94 peptide (specific to HCC cells), 2) YSK05 lipid (increased cytotoxic effects and limited endosomal escape), and 3) specific phosphatidylcholine/cholesterol ratio (improves liposome stability). 44They demonstrated both in vitro and in vivo that their nanoliposome had specific uptake to HCC cells over normal hepatocytes, potentiated sorafenib's effects, and resulted in profound tumor regressions (~70%). 44,45dditionally, Woitok et al. delivered siRNA targeting Jun N-terminal kinase-2 (Jnk2), known to affect fibrosis progression, in lipid nanovesicle to mice with chronic liver disease and demonstrated decreased HCC premalignant nodules and a shift in the immune microenvironment of the diseased liver. 46Moreover, targeting key cellular pathways in HCC with siRNAs has also been feasible as demonstrated by the work from Fitamant and colleagues. 47They delivered nanovesicles containing siRNA to Yes-associated protein 1 (YAP), a key downstream transcriptional co-activator of Hippo signaling, resulting in tumor regression through directing hepatocyte differentiation to normal hepatocyte-like cells.Other groups have also delivered nanoliposomes containing siRNAs targeting PD-L1, 48 T cell immunoglobulin mucin-3 49 (Tim- 3; immune checkpoint molecule), vascular endothelial growth factor 50 (VEGF; angiogenic factor), alpha-fetoprotein 51 (AFP; biomarker for HCC), cyclo-oxygenase-2 52 (COX-2; important for prostaglandin synthesis in inflammatory processes), hypoxia inducible factor 1 subunit alpha 53 (HIF1a), or RNA N 6− methyladenosine (m 6 A) reader protein YTHDF1 54 either alone or in combination with chemotherapeutics.Moreover, miRNAs can be packaged into nanoliposomes to target specific cellular pathways.For example, Zhao et al. loaded miR-375 and sorafenib in nanoliposomes to hinder autophagic processes and reduce tumor burden. 55Lastly, mRNAs may also be packaged into nanovesicles for HCC therapy.Lai et al. demonstrated that delivery of IL-12 mRNA in nanovesicles reduced tumor burden and prolonged survival of transgenic MYCinduced HCC mice. 56This effect was also associated with a shift toward a more anti-tumor immune microenvironment with increases in T helper cells and IFNγ expression. 56Similar effects were seen with mRNA for OX40L encapsulated nanovesicles. 57Overall, lipid nanoparticles provide an efficient platform to deliver both chemotherapeutics and gene therapy at subtoxic doses with high efficiency and stability. 44,53s previously discussed, modifying the outer shell of the nanoliposome can improve the delivery efficiency and targeting to the desired end organ.For targeting HCC cells specifically, various groups have functionalized nanoliposomes to target CXCR4 high expressing cells given its sorafenib resistance mechanisms.9][60] Additionally, GalNAcconjugated nanovesicles have demonstrated considerable success in highly relevant animal models of molecular subsets of HCC with the nanoliposomes encapsulating siRNAs to oncogenic drivers, such as CTNNB1. 61,62Also, the lipid configuration and inclusion of PEG/mannose into the membrane can also affect targeting to different liver cell types. 63herefore, using targeting molecules on nanoliposome surface can improve the efficiency of tumor cell transfection and diminish off-target effects.
Moreover, another strategy is modifying the lipid composition of the liposome for controllable release of chemotherapeutic agents through stimuli responses.Examples of this include using either temperature sensitive, 64 pH responsive, 65,66 photosensitive, magnetic-sensitive, or ultrasound-guided lipids. 67In terms of temperature-sensitive lipids, Peng et al. 64 utilized PF127 (copolymer) which has temperature-sensitive properties and aids in degrading the nanoliposome following photothermal conversion of IR-780 (a near-infrared [NIR] dye) also contained on the nanoliposome surface.This combination of PF127 and IR-780 allowed for efficient doxorubicin and sorafenib release at the tumor site in vivo.Also, as illustrated by Li et al., 65 interchanging the nanoliposome bilayer to include the cationic lipid (2E)-4-(dioleostearin)-amino -4-carbonyl-2-butenonic (DC), can allow for direct tumor cell internalization upon conformational change in the acidic tumor microenvironment, and subsequently release its cargo in the acidified endosome.This allowed for reduced drug toxicity and targeting of tumor cells over normal hepatocytes.Overall, the lipid composition can allow for improved pharmacokinetics and tumor cell internalization.

Extracellular vesicle-based drug delivery platforms for HCC
Extracellular vesicles (EVs) are lipid nanovesicles (50 nm to >2000 nm) which are spontaneously produced by nearly all mammalian cells and released into extracellular fluid as part of autocrine, paracrine, and endocrine cell-to-cell signaling circuits. 68There are various EV subclasses, including exosomes (derived from endosomal membrane trafficking machinery), microvesicles (outward plasma membrane blebbings), and apoptotic bodies (from apoptotic processes).All EVs contain cargos comprising various membrane and soluble proteins, nucleic acid species, and metabolites, which are specific to their cell of origin.Once released into the extracellular milieu, EVs travel systemically until they make contact with and fuse with their target cell plasma membrane through various endocytic or phagocytic mechanisms. 69he natural ability for EVs to avoid immune system clearance, systemically travel to end organs, and package cargos within lipid bilayers has made them an attractive tool for drug delivery.Through the use of nanomedicine platforms, EV mimetics are being translated to the clinic as novel drug delivery vehicles.Various researchers have developed different EV mimetic technologies, either through modifying parental cells (e.g., stem cells, fibroblasts, immune cells) and isolating their EVs for delivery, or ex vivo loading of cargo components into EVs.][72][73][74] The main class of EV mimetics utilized for HCC targeted therapy are siRNA-encapsulated EVs, which target specific mRNAs encoding oncogenic signaling proteins.Various groups have identified target genes, which when suppressed, may synergize with ICIs.One target is CD38, a transmembrane protein which is aberrantly expressed in many tumors and associated with a pro-inflammatory tumor microenvironment, and has been shown to be associated ICI response. 75,76EVs isolated from bone marrow mesenchymal stem cells packaged with siRNA to CD38 (via electroporation) reduced HCC tumor burden, metastatic potential, repolarized macrophages from M2 (immunosuppressive) to M1 (proinflammatory) phenotype, and improved ICI response. 75Other genes/pathways identified which have been targeted with siRNAs packaged in EVs, include components of the ferroptosis pathway (GPX4 and DHODH), 77 cell cycle regulation (CDK1), 78 JAK/STAT pathway (STAT6), 79 and NFkB pathway (p50 subunit). 80Rather than directly targeting translation of molecules displayed on tumor cell surface mediating immunosuppression, another approach is targeting the underlying genetic mutation of the tumor cell.Matusda and colleagues designed an siRNA targeting CTNNB1 delivered within EVs. 81 Using the Met/β-catenin mouse model (which represents ~ 10% of human HCC), they remarkably demonstrated that delivery of milk-derived EVs encapsulating siRNA to CTNNB1 (using transfection techniques) reduced tumor burden, in part through reversing the immunosuppressive tumor microenvironment driven by β-catenin, which allowed for synergy with ICIs.Another group utilized a similar platform, but functionalized the EVs to target EpCAM-positive HCC cells. 82These studies along with others previously mentioned 61,62,82 provide direct evidence that therapeutically targeting oncogenic mutations with siRNAs are effective approaches to treat HCC.][85] Similar to siRNAs, miRNAs packaged into EVs offer another platform to target actively proliferating cancer cells.Many miRNAs have been implicated in HCC pathogenesis, including miR-21, miR-125b, miR-155, and miR-221/222. 86Particularly, miR-125b down-regulation is associated with worse overall survival. 87Baldari and colleagues isolated EVs (via polymer-based methods) from adipose-derived stromal cells (ADSCs) engineered to express miR-125b with a unique "ExoMotif" sequence that increases release of miR-125b into EVs. 88These EVs were delivered in vitro to HepG2 and HuH-7 cells and reduced cell proliferation, along with expression of p53 signaling pathway components. 88In another study, Mahati and colleagues loaded mesenchymal stem cell (MSC)-derived EVs with miR-26a (via electroporation) and observed impaired cell proliferation and migration in vitro, along with reduced tumor burden in subcutaneous HCC models. 89Lastly, Ellipilli and colleagues demonstrated that combined Paclitaxel and miR-122 (liver specific miRNA; reduced levels shown in HCC) administration within GalNAc-EVs reduced tumor burden in multiple mice xenograft HCC models. 90Complementary to RNAi, another strategy for EV therapeutics includes exogenous or endogenous small molecule and protein loading.][93][94] Monoclonal antibodies, nanobodies, and various cytokines can even be packaged into EVs to target specific immune checkpoint molecules to induce native immune activity. 95,96However, these techniques may damage the membrane integrity of EVs. 92,97ndogenous protein loading into EVs is a novel technique which hijacks cell signaling cascades to load particular payloads into EVs, which can be isolated, and subsequently administered as therapeutics.Different groups have utilized the ability of FK506 binding protein (FKBP) and FKBP12-rapamycin-binding (FRB) domain to heterodimerize following rapamycin administration. 98,99The FRB domain is fused to the protein of interest via a GGSGG linker, and the FKBP domain is fused to a canonical EV protein (e.g., CD81 or CD63) via the N-myristoylation sequence to facilitate protein entry into EVs.Cell lines can be modified to express these fusion proteins and EVs can be isolated and delivered in vivo for effective protein delivery. 98,99Small molecule/chemotherapeutic agent packaging into EVs have demonstrated potential, including the use of doxorubicin, 100 norcantharidin, 101 and sorafenib. 1024][105] Overall, these methods of protein/small molecule packaging are appealing options for therapeutic delivery to liver.
In the last two decades, recombinant Adenoassociated viruses (AAVs) have been explored as gene delivery vehicles for cancer due to their ability to target many cell types and long-lasting gene expression. 106More recently, EVs have been shown to be associate with isolated AAVs (termed "vexosomes") during virus isolation from cellculture media.These vexosomes have become an alternate gene delivery vehicle. 107,108Moreover, vexosomes protect AAVs from antibody neutralization, a major issue for AAV in vivo translation. 109han et al. isolated AAV6-derived vexosomes (via ultracentrifugation) containing an inducible caspase 9 (iCasp9), which upon delivery with a prodrug (AP20187), results in impaired HCC cell proliferation in vitro and tumor cell death in vivo via apoptosis. 110Overall, vexosomes are another gene therapy-based EV mimetic technology which are highly efficient delivery vehicles, require lower therapeutic doses than AAVs, and are not cumbersome to manufacture.
Lastly, EVs isolated from allogeneic or autologous cell sources are another therapeutic option for HCC.Kim and colleagues have demonstrated that EVs isolated from natural killer (NK) cells, which contain proteins important for mediating immunogenic cell death, can functionally impair HCC growth in vitro and in vivo. 111These NK-EVs (isolated via ultracentrifugation) express granzyme B, FasL, and TRAIL and mediate apoptosis through inducing caspase-3, 7, 8, and 9 upon internalization in tumor cells.Additionally, another cell type with promise as a therapeutic source of EVs are ADSCs.Wu and colleagues revealed that ADSC-EVs (isolated via ultracentrifugation of culture media) decreased hepatic fibrosis and glutamine synthetase levels, suggesting that this may have therapeutic potential in subsets of HCC. 112Moreover, another cell type which has demonstrated promise are dendritic cell (DC)derived EVs.The pathogenesis of CTNNB1mutated HCC involves defective recruitment of DCs, 113 likely making DC-EVs an interesting platform as an HCC therapeutic.Lu and colleagues systemically administered DC-EVs in three different HCC models and observed shifts in the tumor microenvironment such as increases in cytotoxic CD8 T-cells and fewer immunosuppressive T regulatory cells, which associated with tumor regression. 114astly, M1 macrophages-derived EVs loaded with docosahexaenoic acid have been shown to induce ferroptosis and reduce tumor burden in orthotopic HCC models. 115Therefore, EVs isolated from allogeneic sources have intrinsic capabilities to alter tumor cell survival and growth.However, autologous-derived EVs may have improved tumor targeting properties.Villa et al. illustrated that EVs derived from blood plasma of cancer patients had selective uptake into associated patient-derived xenograft (PDX) mouse models. 116Therefore, autologous EV sources may be another pipeline for manufacture with improved tumor-specific targeting properties.

Challenges in good manufacturing practices for nanovesicle therapeutics
Many of the challenges of translating nanovesicle therapeutics are shared between synthetic and natural platforms; however, this section will focus on the nuances associated with translating EV-based therapeutics.The first consideration is isolation purity.Current clinical Good Manufacturing Processes (GMP) of therapeutic EVs may lead to downstream isolation of contaminants (e.g., viral) from cell culture supernatants. 117For regulatory agency approval of EVs, a complete biochemical characterization is required for biologics, which remains incomplete due to technological limitations and EV isolation best practices. 117Additionally, given EVs are a cell-free therapy, the mechanisms of cellular uptake/targeting, cargo delivery/release, and an understanding of the precise bioactive and nonactive components are unclear. 117,118hether the membrane lipids/proteins, or the proteins/nucleic acids in the lumen, or both, contribute to the intended therapeutic effect is not determined.Therefore, extensive functional assays, "-omic," and imaging platforms are needed to fully elucidate and differentiate the physiochemical properties and bioactivity of EVs.The International Society for Extracellular Vesicles (ISEV) has established guidelines for clinical GMP of therapeutic EVs. 119he second consideration is cellular source and cell culture ecosystems of therapeutic EVs.As discussed in the previous section, cellular sources of therapeutic EVs for cancer can include either stem cells, immune cells, or nonparenchymal/stromal cells.Each of these cell types require different culture methods and release differing quantities of EVs.Additionally, cell culture practices of these cell types typically include utilizing fetal bovine serum (FBS) as a culture media supplement, which presents challenges due to introducing FBS-derived EVs into the pool of cell culture-derived EVs. 120Simply, this contamination means that upon isolation of EVs from the cell culture supernatant, the final EV fraction will contain both EVs from the FBS and the cultured cells. 120To circumvent these issues, the use of EV-depleted FBS or serum-free culture conditions have been proposed, with each providing their own inherent limitations, including cell death, incomplete elimination of FBS-derived EVs, and changes to cellular differentiation/state. 120,121oreover, when culturing cells, the passage number, cell seeding density, and timing of media harvest can contribute to heterogeneity in cultured cells, and thus EVs isolated. 117,122he third consideration is the scale of manufacturing.For mass production of EVs, unique culture systems are needed, such as stacked culture vessels or bioreactors. 117,118Also, each EV isolation protocol (e.g., ultracentrifugation, precipitation, sizeexclusion chromatography, and filtration) present differences in efficiency, quantity, purity, and quality of final EV formulations. 123For example, although centrifugation-based approaches improve EV purity, this is at the expense of cost and time. 120Lastly, with high-volume manufacturing, evaluating differences in batches is also important to consider. 117

Oncology clinical trials implementing nanovesicle platforms
The translation of lipid nanoparticles and EVs to clinical practice as HCC therapies has not moved swiftly.Currently, EVs are being studied as diagnostic biomarkers 124 for HCC to detect initial diagnosis, response to therapy, and disease recurrence 125 using DNA mutations 126,127 or methylation 128 patterns, mRNA 129 /miRNA signatures, 130 or proteins 131,132 encapsulated in their lumen.This section will briefly cover inhuman studies in oncology which has successfully translated nanovesicle therapeutic platforms to the clinic.To investigate whether lipid nanovesicles were actively being translated into clinical trials, we surveyed the clinicaltrials.govwebsite to search for active or terminated trials.A review of the clinicaltrials.govwebsite for clinical trials related to "cancer" and "exosomes" yielded 132 studies, with 7 unique studies focusing on therapeutic applications (Table 1).Additionally, a review for clinical trials related to "cancer" and "nanovesicle" yielded 12 studies, with 7 unique studies focusing on therapeutic applications (Table 1).Overall, there are few trials investigating the therapeutic potential of lipid nanovesicle platforms in HCC space.Notably, Omega Therapeutics is leading their phase I/II MYCHELANGELO™ trial (NCT05497453) evaluating OTX-2002 as monotherapy or in combination with HCC standard of care (TKIs or ICIs), which is an mRNA therapeutic encapsulated in lipid nanovesicle which decreases c-MYC gene expression through modifying the c-Myc transcript via epigenetic modulation. 133hey most recently (September 2023) have described preliminary results in 8 patients and observed on-target effects with associated decreases in c-MYC gene expression. 134This signals the transition of siRNA/mRNA lipid nanovesicle therapeutics from the preclinical to clinical realm to target traditionally "undruggable" oncogenic drivers to be used in conjunction with standard of care agents (i.e., TKIs or ICIs).

Conclusions and future perspectives
Lipid nanovesicles are next-generation drug delivery vehicles swiftly becoming part of the oncologist armamentarium.Compared to the administration of "naked" drug, encapsulated drug within lipid nanovesicles allows for reduced toxicity, improved biocompatibility, and improved in vivo efficacy through enhanced delivery to end-organ and target cell internalization.Several studies have illuminated the potential of lipid nanovesicles, both synthetic and natural, as drug delivery platforms in preclinical models and in patients, with several companies licensing these technologies from academia and translating their products to the clinic.These platforms are ideal drug delivery vehicles for treating various liver pathologies, including cancer, due to the liver's inherent dual blood supply and fenestrated endothelium to allow for efficient systemic administration and hepatocyte delivery, respectively.Also, these nanovesicles are opsonized by ApoE and recognized by the hepatocyte LDLR for efficient targeting.Or functionalization of the nanovesicle may allow for directed cell-type specificity. 135here are distinct advantages and disadvantages of each platform (Table 2).To improve the translation of this new EV class of biologics to the clinic, there are several technical challenges, including improving isolation techniques, component characterization, and manufacturing. 70,117Additionally, an enhanced understanding of the factors lending toward high biocompatibility of EVs may augment the development and translation of synthetic nanovesicles. 117Despite these challenges, the future is bright for nanovesicle therapeutic applications in oncology, particularly EVs, and as technology advances, these roadblocks will only become surpassed and push these biologics toward clinical practice.For translation of EV therapeutics, lessons may be learned from some of the hurdles overcome by those involved in translating nanoliposome formulations. 136For example, for nanoliposomes, great detail was undertaken to understand how the composition of ionizable lipids, various active drug loading techniques, and the cholesterol composition in the membrane affected drug stability, and thus enhanced in vivo activity. 137Additionally, the size of the nanovesicle plays an important role in the ability to target the liver (and specific cell-type), with studies concluding <100 nm is ideal for hepatocyte delivery. 136,138Interrogation of all these different tunable characteristics of nanovesicles for EVbased drug delivery vehicles will ultimately improve their translatability to the clinic.

Figure 1 .
Figure 1.Schematic representation of nanoliposome and extracellular vesicle loading strategies, cellular uptake mechanisms of these drug delivery vehicles, and clinical parameters to monitor for toxicity in patients.Figure made in BioRender.

Table 1 .
Clinical trials registered on clinicaltrials.Gov website for use of lipid nanovesicles and extracellular vesicles in oncology.